Post-transcriptional regulation of messenger RNA (mRNA) stability and translation is important for controlling gene expression. This project seeks to generate and utilize structural information to enhance our understanding of these processes. RNA silencing, the destruction of mRNA by double stranded RNA containing corresponding sequences, has proven to be a powerful tool to knock out expression of target genes in eukaryotic cells and may have therapeutic potential. In the past few years, much has been learned about the mechanism by which RNA silencing occurs, including the identification of proteins involved in the process. PUF proteins are regulators of gene expression important for embryonic development and germline stem cell maintenance.[unreadable] [unreadable] This project has two main focuses. The first is to study proteins that suppress RNA silencing or are involved in the RNA silencing pathway. We are using structural and biochemical methods to understand their functions.[unreadable] [unreadable] The second is to study PUF family proteins. We have determined the crystal structure of human Pumilio 1 protein alone and in complex with RNA. These structures have provided rules for sequence-specific RNA recognition by this family of proteins. We are using this information to create RNA-binding proteins with altered RNA sequence specificity. We are also studying Puf proteins from different organisms to more fully understand the structure, RNA recognition, and post-transcriptional gene regulation by this family of proteins.